Lipidspot

For 5hmC detection, cells were fixed with 4% paraformaldehyde for 20 min, for BGLAP detection, cells were fixed in ice-cold methanol at −20 °C for 10 min. Cells were then permeabilized with 0.5% Triton in 4% paraformaldehyde for 5 min at room temperature. For 5hmC detection, chromatin was denatured with 2 N HCl for 20 min and then washed twice with 100 mM Tris-HCl pH 8.0 for 5 min. Blocking was performed in 1% donkey serum (Abcam, ab7475) for 1 h. Cells were then incubated with the selected antibodies (5hmC, Active Motif, #39769, 1:5,000; BGLAP, Abcam, ab13421, 10 µg/ml) or normal rabbit IgG (Santa Cruz, #sc-2027, 1:100) in 1% donkey serum in PBS overnight and then with an appropriate secondary antibody (goat anti-rabbit Alexa-647 or goat anti-mouse Alexa-488, Thermo Fisher; A-21245 and A-11001, respectively) diluted 1:400 in 1% donkey serum in PBS for 1 h. Adipogenic lipid droplet formation was visualized with LipidSpot (Biotium) as per manufacturer’s recommendations. Image acquisition and analysis was performed with a Zeiss LSM 780 microscope or a Zeiss Axio Vert.A1 microscope in conjunction with Zen blue edition software version 2.3 or Zen black edition software version 14.0 as well as ImageJ software (National Institute of Health, Bethesda, MD).

After lipid incubation, coverslips were lifted from the wells and cells were fixed for 15 min in 4% PFA, washed 1× with PBS, and incubated for 30 min in 1:1000 LipidSpot (Biotium, Fremont, CA, USA). Coverslips were then mounted onto slides using Fluoroshield mounting media with DAPI (Abcam). Slides were kept at 4 °C. Wide field images were obtained on a Nikon Ti2 microscope using a 20× objective. Single cell images were obtained on a Nikon A1R Laser Scanning Confocal Microscope (Nikon, Tokyo, Japan) using an oil immersed 100× objective. Astrocytes were selected in the 405 channel (DAPI-stained nuclei) and then the 610 channel was added (LipidSpot-stained LDs) and the images were captured. Z-stacks were taken in a range of 10 µM, 40 images per stack. 3-D reconstructions were processed in Imaris 9.2 software using the surfaces module to obtain statistics for lipid droplets. Experimenter was blind to slide identity and slides were processed in random order under identical microscope settings.

Liver tissues were fixed in 10% (v/v) neutral buffered formalin solution, embedded in paraffin, and cut at 8 μm. Tissue sections were then stained with hematoxylin and eosin to visualize histological changes. Intracellular lipid droplets were visualized under an Olympus light microscope after Oil Red O or LipidSpot (70065, Biotium) staining of frozen liver sections that were embedded in the Tissue-Tek Optimum Cutting Temperature (OCT) compound. At least 3 images per specimen were obtained for analysis.